title: An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier.
abstract: Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia.
authors.affiliation: Department of Molecular and Cellular Physiology and 2 Department of Chemical Engineering, Stanford University School of Medicine, Stanford, CA 94305.
authors.forename: David K
authors.initials: DK
authors.lastname: Breslow
authors.forename: Elena F
authors.initials: EF
authors.lastname: Koslover
authors.forename: Federica
authors.initials: F
authors.lastname: Seydel
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Spakowitz
authors.forename: Maxence V
authors.initials: MV
authors.lastname: Nachury
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002923:Cilia / Q000378:metabolism*; D004058:Diffusion; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008954:Models, Biological; D008970:Molecular Weight; D022022:Nuclear Pore / Q000378:metabolism; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013997:Time Factors; D059008:Time-Lapse Imaging; D014162:Transfection
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins